GSM7658540: Cut&Run on IHA-7d-Dox using H3K9ac antibody; Homo sapiens; OTHER
Cut&Run assay was done as described in (Meers et al., 2019; Skene and Henikoff, 2017) with slight modifications as follows: Cells were harvested and counted, with 200,000 cells taken per reaction. Permeabilized cells, bound to Concanavalin A-coated beads (Bangs Laboratories, BP531), were mixed with individual primary antibody (diluted 1:150-1:200) and incubated overnight at 4°C while rotating. Secondary antibody anti-rabbit HRP was used as a negative control. pAG-MNase enzyme (generated in the Department of Life Sciences Core Facilities, WIS, using Addgene plasmid 123461) was added to each sample followed by incubation step of 1 h at 4°C. Targeted digestion was done by 15 min incubation on ice block (0°C) under low salt conditions. DNA purification was done using Nucleospin gel and PCR clean-up kit (Machery-Nagel, 740609). Libraries were prepared from 1-20ng of DNA as previously described [61]. Briefly, DNA Fragments were repaired by T4 DNA polymerase (NEB, M0203), and T4 polynucleotide kinase (T4 PNK, NEB, M0201) was used to add a phosphate group at the 5′ ends. An adenosine base was then added by Klenow fragments (NEB, M0212) to allow efficient ligation, using T4 quick ligase (NEB, M2200), of sequencing adapters, which contain a T-overhang. DNA fragments were amplified by PCR reaction (Pfu Ultra II fusion, Agilent Technologies, 600670), which also introduced the Illumina-P5 adapter at one end of the molecule. SPRI beads were used to purify proper sized DNA fragments after each enzymatic step. Libraries were quantified by Qubit (ThermoFisher Scientific) and proper fragment range (200-400bp) was verified by TapeStation (Agilent). Sequencing was done on a Next-Seq 500 instrument (Illumina) using a Nextseq 150 cycles mid output kit, allocating 10M reads per sample (paired end sequencing).